重组蓖麻毒素A链表达及其与天然蓖麻毒素毒性比较研究

本试验旨在制备有高生物活性的重组蓖麻毒素A链(r-RTA)并与天然蓖麻毒素(n-RT)进行毒性比较.根据NCBI公布的RTA序列合成基因片段,并将其克隆于pET-28a载体后,利用大肠杆菌进行原核表达,镍柱亲和层析纯化上清,500 mmol/L咪唑溶液洗脱获得可溶性r-RTA蛋白,通过SDS-PAGE及Western blotting对其进行鉴定并用ELISA测定其免疫原性后,对r-RTA与n-RT进行动物和细胞试验毒性比较研究.结果显示,该r-RTA在上清中表达率为31.2%;每升细菌培养物纯化后可得20 mg目的蛋白,纯度≥90%,大小为32 ku.其免疫原性约为n-RT的1.27倍;通过获取的n-RT细胞半抑制浓度(IC₅₀为0.01 μg/mL)及动物半数致死量(LD₅₀为(3.27±0.44) μg/kg),以相同浓度进行细胞感染和动物攻毒试验,结果显示,同等剂量下,在细胞试验中,n-RT毒力是r-RTA的2 700倍;在动物试验中,n-RT组对动物致死率为40%,r-RTA组动物无死亡.结果表明,单独RTA,在没有RTB协助下,具有一定的毒性作用,但毒力将显著降低.该结果将为开发基于RTA的蓖麻毒素疫苗提供重要数据和理论支撑.     

碱性磷酸酯酶基因在鸡胚和人乳腺癌细胞中的表达

通过PCR方法从pSEAP2-control质粒获得人胎盘碱性磷酸酯酶(PLAP)基因,并克隆到鸡输卵管特异性表达载体(pAB-Sig-SV40)卵清蛋白基因调控区下游.pSEAP2-contro1和pAB-Sig-AP-SV40质粒转染鸡胚细胞和乳腺癌细胞(MCF-7),转染48h后以对硝基苯磷酸钠(pNPP)为底物检测PLAP的活性,结果pSEAP2-contro1在鸡胚细胞和MCF-7细胞中表达PLAP,pAB-Sig-AP-SV40在鸡胚细胞中不表达,而在MCF-7细胞中表达.说明鸡胚细胞可以表达有活性的人源糖蛋白,另外也说明在雌激素受体细胞中,鸡卵清蛋白基因启动子可启动外源基因表达.     

抗蓖麻毒素单链抗体基因的构建及其在大肠杆菌中的表达

为构建和表达抗RT单链抗体(ScFv)蛋白,用RT-PCR方法从能分泌特异性抗RT单克隆抗体(McAb)的杂交瘤细胞中分离纯化抗体VH和VL基因。用重叠延伸PCR方法将VH和VL拼接在一起,构建抗RT-ScFv基因。将ScFv基因连接到pMAL-p2X表达载体,转化TB1表达菌。阳性克隆用IPTG诱导18h,Western blotting鉴定重组蛋白。结果表明,试验成功扩增出了ScFv基因,长度约为750bp。通过DNA序列测定和分析,构建出VL-(Gly4Ser)3-VH。其VH全长363bp,可编码121个氨基酸,VL全长324bp,可编码108个氨基酸。SDS-PAGE和Western blotting分析结果表明,抗RT-ScFv在TB1表达菌中获得高效表达,pMAL-p2X表达的ScFv加上同时融合表达的MBP标签分子质量约为75ku。本试验成功构建了pMAL-RT-ScFv表达载体,并获得了高效表达。     

人胰岛素基因杆状病毒表达载体的构建及其在Sf9细胞中的表达

对人胰岛素基因真核表达载体（pCMA／mINS）进行Xho I酶切，回收的含人胰岛素基因组基因的1608bp片段与线性化的杆状病载体pFast Bac I进行连接，获得重组载体pFast/mINS。将该重组载体转化DH10Bac感受态细菌，在体内进行重组，并经2次抗性与蓝白斑筛选，得到杆状病毒重组载体Bacmid/mINS。将该载体转染Sf9细胞，获得了重组杆状病毒，经Tricine-SDS-PAGE和Western-blotting检测，重组杆状病毒在Sf9细胞中的表达产物是胰岛素原，而细胞培养上清中未检测到胰岛素和胰岛素原。     

猪生长激素基因在昆虫细胞中的分泌表达

将PCR将增得到pGH基因插入到带有polh启动子和gp67强信号肽序列的杆状病毒转移载体pAcGP67-A中，构建重组质粒pGP67pGH，并与线性化致死缺失型苜蓿丫纹夜蛾核多角体病毒（AcMNPV-OCC)基因组DNA共转染Sf9细胞，构建出重组病毒AcMNPV-pGP67-pGH-OC^-。感染重组病毒的Hi5细胞的表达产物的SDS-PAGE和Western blot结果表明，细胞可溶蛋白和培养液上清中均有一条分子量约为22kDa的猪生长激素特异性反应带，且培养液上清中的目的蛋白分子量比天然pGH略大一些，薄层扫描仪扫描估测可知，重组pGH分别占细胞可溶蛋白的8．98％和培养液上清总蛋白的3.61%。将表达96h的培养液上清浓缩液进行N－糖基化分析，结果显示重组pGH无N－糖基化加工修饰。     

人血小板因子Ⅳ在家蚕杆状病毒表达载体系统中的表达

将人血小板因子Ⅳ (HumanPlateletFactorⅣ ,简称hPF4)基因重组于家蚕杆状病毒转移载体pBacPAK8中 ,获得重组转移载体pBacPAK PF4,并与线性化病毒Bm BacPAK6DNA共转染家蚕培养细胞 ,在细胞内发生同源重组 ,获得重组病毒BacPAK PF4。Southern杂交结果表明重组病毒基因组中含有hPF4基因。重组病毒以MOI=10感染家蚕培养细胞 (2× 10 6个细胞 )和家蚕 5龄幼虫 ,表达产物用体外培养的血管内皮细胞测定其生物活性 ,测得表达量在家蚕培养细胞中第 3天达到最高值为 6 0 88μg/ 2× 10 6个细胞 ;在蚕体内表达第 5天达到最高值 ,表达量明显高于家蚕培养细胞     

中短链脂肪酸在无抗饲料中的应用

在现代畜牧业中,为了预防动物疾病和提高生产性能,普遍在饲料中使用抗生素。由于抗生素在畜牧业中的过度使用导致细菌耐药性的产生和抗生素的残留,严重影响到人类和畜禽的健康。因此,迫切地需要制定策略来替代抗生素用于生产食品的动物,特别是畜禽。中短链脂肪酸作为安全高效的添加剂已经畜牧生产中得到广泛的应用,文章以无抗饲料为背景,论述了中短链脂肪酸的研究进展和展望,为无抗饲料开发及使用提供参考。     

AsiaⅠ型口蹄疫双效表达载体在BHK-21细胞中的表达

将构建好的双向反义表达载体PQC-AS-P12A3C经脂质体2000转染Ampho-pack293细胞,收获假病毒,在聚凝胺的介导下将其转染到BHK-21细胞,一定时间后进行检测。通过荧光抗体染色、目的基因的整合鉴定和RT-PCR检测等表明,目的基因P12A在细胞中成功表达,且已经整合到细胞染色体基因组中,病毒抑制试验则表明重组反义质粒对FMDV的产生起到一定的抑制作用。     

猪RUVBL2真核表达载体的构建及其在CHO细胞中的表达

RUVBL2(RuvB-like 2)蛋白是进化上高度保守AAA+(ATPases Associated With Diverse Cellular Activities,AAA)家族成员之一,CHO(Chinese Hamster Ovary)细胞被广泛地用于表达重组DNA蛋白。为探究猪睾丸组织的RUVBL2基因是否能够在CHO细胞中表达,本实验以猪睾丸组织的cDNA为模板,PCR扩增RUVBL2目的基因,并将其克隆至pIRES2-EGFP(Mammalian Expression Vectors pIRES2-EGFP)载体上,进一步转化到DH5α中,再进行PCR、酶切及测序鉴定;将重组质粒转染到CHO细胞中,再进行荧光、RT-PCR、Western blot检测。结果显示:PCR、酶切及测序都证实了RUVBL2基因正确地插入到了载体质粒pIRES2-EGFP的多克隆位点;荧光、RT-PCR、Western blot也证实了RUVBL2基因在CHO细胞中的成功表达。本实验成功构建了猪的pIRES2-EGFP-RUVBL2真核表达载体,并证实了猪睾丸组织中的RUVBL2基因能够在CHO细胞中表达。     

小反刍兽疫病毒N基因在昆虫细胞中的表达

为了研发小反刍兽疫病毒ELISA检测试剂盒中替代全病毒的抗原物质,参照GenBank公布的小反刍兽疫疫苗株Nigeria75/1的全基因组序列(GenBank登录号:X74443),人工合成表达核蛋白的N基因开放阅读框序列,通过PCR扩增、经引物设计引入的EcoRⅠ和KpnⅠ特异性酶切位点,将N基因克隆于昆虫杆状病毒表...     

Expression of p63 in the mouse primordial germ cells

Proteins encoded by p63 gene a have structural similarity with tumor suppressor p53, and were thought to induce cell cycle arrest and apoptosis during development. The p63 proteins are also expressed in the basal cells of many epithelial tissues in the adult, and supposed to play important roles in maintaining the epidermal stem cells. Previously, we reported the p63 expression in the testis of mouse embryos, suggesting their involvement in the growth arrest and apoptosis of testicular germ cells (Nakamuta and Kobayashi, J. Vet. Med. Sci. 65:853-856). In this study, we investigated the timing of this p63 expression in the germ cells during migration and colonization to the gonads. Immunohistochemical analysis of mice from embryonic day (E) 7.5 to E12.5 demonstrated that p63 positive reactivity was seen as early as E8.5 when the founder cells of germ cells, primordial germ cells (PGCs), were located in the hind gut epithelium, but PGCs were negative for p63 at E7.5 when they first appeared. p63 is expressed as six isoforms, resulting from alternative splicing at C-terminus and by the use of two promoters that generate variations at N-terminal end. RT-PCR analyses suggested that different types of p63 mRNAs were likely to be expressed in PGCs during development. These results imply that p63 may be involved in the regulation of PGC development by controlling the gene expression required for their migration and colonization to the gonads.     

用麦胚芽粉取代对虾饲料中鱼粉的可行性研究

鱼粉是水产饲料的重要优质蛋白源。然而,日益短缺和价格攀升的鱼粉已成为制约水产养殖业的瓶颈。因此,寻找可部分代替鱼粉的饲料蛋白源对维持水产养殖业的稳步发展,是十分迫切的,并具有重要的经济意义。小麦胚芽粉是小麦加工副产品,仅占小麦的2%,但含有丰富的可消化蛋白质,其粗蛋白质含量为31%～35%,必需氨基酸的组成优于一般谷物。麦胚芽的脂肪含量约为8%～10%,主要为不饱和脂肪酸,其中18:2ｎ-6约占50%。其脂肪中维生素Ｅ含量每100克高达250～520ｍｇ。维生素Ｅ对水产动物不仅具有重要的营养作用,在饲料中作为抗氧化剂,对防止饲料中脂肪酸…     

Effects of the oviduct and wheat germ agglutinin on enzymatic digestion of porcine zona pellucidae

Seventy-two crossbred gilts were utilized to examine whether the oviduct rendered zona pellucidae resistant to protease digestion, whether the uterus reversed this resistance and whether such a uterine reversal was necessary for hatching. Oocytes were aspirated from follicles 22 to 28 h after onset of estrus (d 0); oviductal and uterine oocytes or embryos were collected on d 1 to 6. These oocytes and embryos were subjected to a solution containing .1% trypsin and .1% pronase (37 degrees C) for observation of zona pellucidae digestion. Zonae of oviductal oocytes were more (P less than .001) resistant to digestion than were follicular oocytes. Placement of follicular oocytes in oviducts for 30 min rendered zona pellucidae more (P less than .001) resistant to protease digestion than oocytes not exposed to oviductal secretions. Resistance of zona pellucidae to proteases, however, decreased (P less than .001) after entry into the uterus. Zonae of morulae retained in oviducts took longer (P less than .001) to digest than those recovered from the uterus. Blastocysts also were treated with wheat germ agglutinin (WGA; 50 micrograms/ml) for 40 min to determine whether artificial induction of zona resistance to enzymatic digestion affected the ability of embryos to hatch. Though WGA treatment delayed (P less than .001) enzymatic digestion of zona pellucidae, time from collection to hatching was not affected. This experiment indicated that the oviduct delayed enzymatic digestion of the zona pellucidae, whereas the uterus reversed this delay. The re-establishment of enzyme susceptibility after uterine entry, however, appeared to be unrelated to the subsequent ability of blastocysts to hatch.     

Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

油菜━冬小麦轮作系统中冬小麦的粮饲兼用性能

为探究油菜(Brassica campestris)-冬小麦(Triticum aestivum)轮作下冬小麦的粮饲兼用性能,在黄土高原塬区,对油菜后茬冬小麦(BW)和休闲后冬小麦(FW)系统中冬小麦‘陇育216’分别进行冬季刈割(Cut1)、春季刈割(Cut2)和不刈割(Uncut)处理,测定了冬小麦生育期水分状况、刈割所得饲草产量和品质以及收获期籽粒产量等。结果表明,1)BW轮作条件下,两次刈割处理下所得饲草产量均高于FW,但处理间饲草的营养指标均无显著差异(P0.05);BW轮作条件下,Cut2所得饲草产量极显著高于Cut1(P0.01),且Cut1和Cut2所得饲草粗蛋白含量均高于20%,相对饲喂价值高于151%,但微量元素含量较低,不能满足家畜的日常需求。2)与FW相比,BW轮作条件下的冬小麦籽粒产量均减少,尤其Cut2导致显著减产20.9%(P0.05),主要表现为单位面积穗数显著减少;BW系统中,Cut1和Cut2籽粒产量均低于Uncut,但无显著差异,且Cut1减产程度大于Cut2。油菜-冬小麦轮作系统中冬小麦粮饲兼用可提供大量优质饲草,而籽粒减产不显著,增加了系统产出,且冬小麦作为饲草利用最佳时间为春季。     

基于APSIM模型的可视化小麦生长系统分析

以气象参数、土壤条件和作物属性等基础数据为驱动,通过耦合基于APSIM的小麦（Triticum aestivum）生长模拟模型、形态结构建成模块构建小麦功能－结构模块（Wheat Function Morphostructure Module）。利用计算机图形技术,在.NET平台上,调用OpenGL作图工具,可以绘制小麦器官三维形态,并通过功能－结构反馈校正建立形态建成过程与生理生态过程的内在融合,分析了小麦形态结构随生长过程动态变化的可视化生长系统的研发过程,从而为提高系统开发中预测的准确性和图形对象高仿真度提供关键的技术支持。     

Pneumonia in mice produced by cell-free extract of cultures of Bordetella parapertussis

A single dose of culture fluid of Bordetella parapertussis freed from cells (CFCF) given intranasally to four-week-old mice free from intercurrent respiratory disease produced a subacute bronchopneumonia, which was similar to that induced by whole cells of ovine isolates of B parapertussis, except that the lesions were less severe and less extensive. From eight hours to 17 days after inoculation, the mice exhibited marked infiltration of neutrophils and macrophages into the alveolar septa, bronchiolar and alveolar spaces, and hyperplasia of peribronchiolar and perivascular lymphoid tissue. Electron microscopy showed damage to ciliated cells, type 1 pneumocytes and alveolar macrophages. These results suggest that extracellular toxic substance(s) produced by ovine isolates of B parapertussis might be involved in the initiation and development of lesions in ovine chronic non-progressive pneumonia.     

Lactic acid bacteria isolated from poultry protect the intestinal epithelial cells of chickens from in vitro wheat germ agglutinin-induced cytotoxicity

1. Poultry fed on wheat-based diets regularly ingest wheat germ agglutinin (WGA) that has toxic effects in vitro on intestinal epithelial cells (IEC) obtained from 14-d-old broilers. Cytotoxicity and the potential role of 14 intestinal bacterial strains in the removal of bound lectins in epithelial cell cultures were investigated.

2. Cytotoxicity was dependent on time and lectin concentration; the lethal dose (LD₅₀) was 8.36 µg/ml for IEC exposed for 2 h to WGA.

3. Complementary sugars to WGA were detected on the surface of one Enterococcus and 9 Lactobacillus strains isolated from poultry. These strains were evaluated as a lectin removal tool for cytotoxicity prevention.

4. Incubation of lactic acid bacteria with WGA before IEC–lectin interaction caused a substantial reduction in the percentage of cell deaths. The protection was attributed to the amount of lectin bound to the bacterial surfaces and was strain-dependent. L. salivarius LET 201 and L. reuteri LET 210 were more efficient than the other lactic acid bacteria assayed.

5. These results provide a basis for the development of probiotic supplements or cell-wall preparations of selected lactic acid bacteria intended to avoid harmful effects of a natural constituent of the grain in wheat-based diets.